| Two-photon Bessel sheet microscopy imaging is a large field-of-view highresolution microscopy imaging method based on two-photon effect and Bessel beam scanning illumination.This method uses a femtosecond pulsed laser in the near-infrared band to perform two-photon excitation of fluorescent material in living organisms with high spatial resolution and illumination depth,and a Bessel light-sheet illumination with large field of view and high temporal resolution,so it has a unique advantage in imaging living organisms with high speed and large field of view for a long time.However,because biological tissues are optical media with inhomogeneous refractive indices,the illumination light sheet is affected by biological aberrations in the process of propagation to the depth of biological tissues,and the fluorescence light-sheet shape diffuses,intensity decreases,and thickness increases significantly.So the illumination field of view cannot reach the ideal size.In order to eliminate the effect of biological aberration on two-photon Bessel light sheet,this thesis applies the adaptive optics technique of direct wavefront detection in the illumination optical path of two-photon Bessel light-sheet microscope and carries out the following research work.1.The theoretical analysis of the Bessel beam generation process using the phase modulation method and the derivation of formulas for the length and thickness of the two-photon Bessel beam are given.Based on the Fresnel diffraction principle,the simulation of the Bessel beam generation process by the phase modulation method is carried out,and the effects of the aperture ratio between the incident beam and the spatial light modulator,and the ratio between the modulation factor and the spatial light modulator pixel size on the modulation effect of the Bessel beam length and thickness are investigated,which provide a strong basis for the selection of the spatial light modulator in the two-photon Bessel light sheet microscope.2.The intensity distribution of two-photon Gaussian beam and two-photon Bessel beam influenced by single Zernike aberration and random aberration of biological tissue was simulated based on Fresnel diffraction principle and Fourier optical analysis.It is found that the Zernike aberration of low azimuthal order(m≤4)is more sensitive to the thickness of the two-photon Gaussian beam,while the thickness of the twophoton Bessel beam is more sensitive to the Zernike aberration of high azimuthal order(n-|m|≤4).Moreover,when the aberration magnitude exceeds 0.3λ,at least 50 Zernike aberrations need to be corrected,which is a guideline for the design and evaluation of adaptive optical systems in two-photon Bessel light-sheet microscopy.3.A method is proposed to combine the use of axicon phase and defocus phase to modulate the length of the Bessel beam and thus achieve the size variation of the isoplanatic region,which causes only 61% of the beam thickness variation compared with the direct change of the modulation factor.Based on the two-dimensional scanning structure consisting of piezoelectric actuator and scanning galvanometer,a two-photon Bessel light-sheet microscopy system with an axial resolution of 600 nm and an illumination field of view of 300 μm×300 μm was designed and built.The functional validity of the two-photon Bessel light-sheet microscope with high-resolution and large field of view was successfully verified.4.The aberration correction algorithm based on the Fresnel diffraction principle is proposed for the problem of the device conjugation relationship between the twophoton Bessel light sheet microscope illumination optical path and the conventional adaptive optics system to simultaneously achieve the generation of Bessel beam and correction of wavefront aberration with one spatial light modulator.A method of direct wavefront detection is proposed by using a spatial light modulator to focus the illumination beam in the sample to excite a two-photon Gaussian fluorescence foci as a fluorescent guide star to address the problem that the wavefront sensor cannot effectively detect the wavefront aberration of a linear-shaped two-photon Bessel beam.This method differs from the conventional method in that no movement or introduction of any other optical components is required,which improves the compactness of the optical system.The intensity and thickness of the aberration-influenced two-photon Bessel beam can be restored to the aberration-free level by numerical simulation and experimental verification.5.Combining the above studies,the overall design of the adaptive two-photon light-sheet microscope system is refined,including optical design,mechanical design and control design,and the completed adaptive two-photon light-sheet microscope system is built.Agar-fixed fluorescent microbeads sample of high concentration is configured for simulating the complex structure of biological sample,and the image scattering lens is placed at the pupil plane of the objective lens for simulating the aberration of biological sample.In this simulation,adaptive correction imaging experiments were performed in the constructed adaptive two-photon Bessel light-sheet microscope system,and the imaging resolution and signal-to-noise ratio are significantly improved after correction.This thesis is the pioneering work of the application of Adaptive Optics in twophoton Bessel light-sheet microscopy.The proposed direct wavefront detection and correction method suitable for the two-photon Bessel microscope provides a reliable technical means for the long-time large field of view and high-resolution imaging of biological tissues. |
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